Characterization of a novel negative regulatory element in the human interleukin 4 promoter

Interleukin 4 (IL-4) is a multifunctional cytokine that plays an important role in hematopoiesis, tumor cell growth, and cellular immune responses. Ex pression of the IL-4 gene is tightly controlled at the level of gene transc ription, and many positive regulatory cis-elements have been identified in the proximal IL-4 promoter region. Relatively little is known about factors that downregulate IL-4 transcription. We performed a detailed deletional a nalysis of the proximal human IL-4 promoter and studied reporter gene activ ity in transiently transfected Jurkat T lymphoblasts. In this report, we ch aracterize a novel negative regulatory element (termed P2 NRE) that is adja cent to a binding site for nuclear factor of activated T cells. Mutation of P2 NRE significantly enhanced the activity of a 175 base pair IL-4 promote r construct in transiently transfected Jurkat T lympho blasts. Using nuclea r extracts from Jurkat cells, we identify a candidate factor (termed Rep-1) that binds uniquely to the P2 NRE in DNA-binding assays. Rep-1 is not rela ted to other factors previously shown to interact with the IL-4 promoter, a nd by UV cross-linking and SDS-PAGE analysis, we found that it migrates wit h a molecular mass of approximately 150 kDa. Characterizing the molecular m echanisms responsible for downregulating the IL-4 promoter should enhance o ur understanding of IL-4-gene dysregulation in disease states.